Background

There are several subtypes of extracellular vesicles (EVs), and among them, exosomes are one of the smallest ones, with a size range between 30 nm and 120 nm (reviewed in Théry et al., 2002). Exosomes contain signature molecules, such as tetraspanins (CD9, CD63, and CD81), and are characterized by specific biogenesis originating in the endosomal pathway (reviewed in Huotari and Helenius, 2011). The EVs can carry and transfer various cargo to other cells, including proteins, metabolites, miRNA, and lipids. One of the exciting research areas is the affinity of exosomes to specific tissues and regions, depending on the characteristics of these EVs. Hence, the biodistribution of EVs should be studied, and EV imaging is required to accomplish this goal. Several methods can be used to track EVs in animal cells and tissues. For instance, bioluminescence imaging (BLI) (Gangadaran et al., 2017) is one of the methods with the highest signal-to-noise ratio, but it suffers from low temporal resolution. Magnetic resonance imaging (MRI) (Busato et al., 2016, 2017) can also be used, but this method is associated with low sensitivity and high operation cost. Finally, fluorescence-based imaging of EVs provides a sensitive method with the highest spatial resolution (Corso et al., 2019). Among the fluorescence-based techniques, GFP protein can be expressed with the lowest penetration, which does not allow for noninvasive in vivo imaging, but has high resolution. Near-infrared (NIR) fluorescence imaging using lipophilic dyes, such as DiR [DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)], can also be used. The two long 18-carbon chains insert into the vesicle membrane avidly, resulting in a negligible dye transfer between EVs. The near IR fluorescent lipophilic carbocyanine DiOC₁₈(7) ('DiR') is weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into membranes. The sulfonate residues incorporated into this DiI analog improve water solubility. Moreover, DiI has an extremely high extinction coefficient and short excited-state lifetimes (~1 nanosecond) in the lipid-rich environment. The DiR shows excitation/emission at 710/760 nm, which, along with its NIR properties, makes this an ideal fluorophore for in vivo EV imaging studies, given the significantly reduced auto-fluorescence from the animal at higher wavelengths (Cook et al., 2015; Somanchi, 2016; Zhao et al., 2021). While there is no perfect method, DiR–based labeling can provide a flexible and low-cost alternative to other methods used for systematic studies of exosomes. This paper describes a technique that uses a lipophilic DiR dye to stain EVs obtained from cell culture, such as cultured macrophages. This approach can be used to track the vesicles for in vitro uptake and in vivo biodistribution studies in animal models.